Method for producing L-sorbosone

ABSTRACT

The present invention relates to a microbiological method for producing L-sorbosone, an important intermediate in the production of vitamin C, from L-sorbosone.

United States Patent Makover et a1.

[451 Oct. 14, 1975 METHOD FOR PRODUCING L-SORBOSONE [75] Inventors: Shraga Makover, Verona; David Louis Pruess, Passaic, both of NJ.

[73] Assignee: Hoffmann-La Roche Inc., Nutley,

[22] Filed: May 6, 1974 [21] Appl. No.: 467,175

[5 6] References Cited UNITED STATES PATENTS 3,043,749 7/1962 Huang 195/47 3,234,105 2/1966 Motizuki et a1 195/47 OTHER PUBLICATIONS Chem. Abstracts, 731R86467z. Chem. Abstracts, 77:1 12489n. Chem. Abstracts, 78:2785121.

Primary Examiner-A. Louis Monacel] Assistant ExaminerThomas G. Wiseman Altorney, Agent, or FirmSamuel L. Welt; Jon S. Saxe; Wi11iam H. Epstein 1 1 ABSTRACT The present invention relates to a microbiological method for producing L-sorbosone, an important intermediate in the production of vitamin C, from L- sorbosone.

6 Claims, N0 Drawings 1 METHOD FOR PRODUCING L-SORBOSONE CROSS REFERENCE TO RELATED APPLICATION This application is related to copending application Ser. No. 285,265, filed Aug.:3l, 1.972, Makover and Pruess. 1

BACKGROUND OF THE INVENTION The compound L-sorbosone, which has the formula:

is disclosed in US. patent application Ser. No. 285,265, filed Aug. 31, 1972, as an intermediate for 2- keto-L-gulonic acid and can be converted'to 2-keto-L- gulonic acid by various enzymatic means. Kondo and Ameyama have disclosed in Reports of Department of Agriculture, Shizueka University, volume 7, page 139 (1957) a pathway from L-sorbose to L-sorbosone utilizing Acetobacter suboxidans. However, it has long been desired to utilize other microorganisms for producing L-sorbosone.

SUMMARY OF THE INVENTION DETAILED DESCRIPTION The novel process of this invention involves the one step microbiological conversion of L-sorbose to L- sorbosone.

Any microorganism of the aforementioned genera can be utilized in accordance with this invention. Among the preferred strains are included: Acinetobacter calcoaceticus (ATCC 10153) Bacillus sp. strain TA (ATCC 27860) Streptomyces cellulocae (ATCC 3313), (Serratia sp. (ATCC 93), Serratia marcescens (ATCC 27857), Aerobacter aerogenes (ATCC 27858), Sarcina lutea (ATCC 9341), Pseudomonas pulida (ATCC 21812), Gluconobatter melanogenus (IFO 3293), Mycobacterium phlei (ATCC 355); and Paecilomyces varioti (ATCC 26820).

The strains of these species are publicly availabe in culture collections, both in the United States and abroad at ATCC (American TypeCultureCollection) Washington, DC. and [F (lnstitutelfor Fermentation) Osaka, Japan. Mutant strains of-the above mentioned microorganisms have superior ability relative to the former wild strains to transform L-sorbose to L- sorbosone. These mutant strains can be. produced from the wild strains by mutagenic treatments. Such mutation can be caused by treating a wild strain with a mutagen such as ultra-violet irradiation, X-ray irradiation or contact with nitrous acid, or by isolating a clone occurring by spontaneous mutation. These means for inducing the desired mutation on a wild type strain may be effected in any of the ways per se well known for this purpose by one skilled in the art. Many of these methods have been described in various publications, for example, Methods in Medical Research Vol. 3, edited by R.W. Gerard, published by the Year Book Publishers, Inc., Chicago, U.S.A., in 1950, and Nature Vol. 183, p. 1829 (1959) by F. Kaudewitz.

The production of L-sorbose is effected by the cultivation of one of the above genera of microorganisms in an aerated deep tank, i.e., under submerged fermentation. The fermentation should be conducted at pH values of from about 5 to about 9 with pH values of from about 6.5 to about 7.5 being preferred. It is particularly preferred to carry out the process of this invention at a pH of from 7.to 7.2. Although the temperature is not critical, best results are. usually obtainedutilizing temperatures of from 20 to 45C., with temperatures of from about 25 to'35C. being particularly preferred. In general, about one to ten days-are required to obtain the bestresults and from about 1 to 2 days are found most suitable. i

The method of the present invention can be carried out by culturing the microorganism in a medium con- .taining appropriate nutrients and L-sorbose. On the other hand, the process of this invention can be carried out by culturingthe microorganisms and then, after culturing, bringing the whole cells or the cell free extract prepared from the culture into contact with L- sorbose. I 1 .5

In the case where the microorganism is cultured in a medium containing L-sorbose and'appropriate nutrients, the microorganism may be cultured in an aqueous medium in an aerated fermentorr The cultivation should be conducted at pH values of from about 5 to about 9, with pHs of from about 6.5 to 7.5 being preferred. Especially preferred is.utilizing a pH of about 7.0 to 7.2. A preferred temperature range for carrying out this cultivation is from about 20.'to about 45C. with temperatures of from about 25 to 30C. being especially preferred. While the time for cultivation varies, with the kind of microorganisms and nutrient medium to be used, about 1 to ,10 days cultivation usually brings about most preferable results. Concentration of L-sorbose in the media varies with the kind of microorganism, is generally desirable to be about 1 to 150 grams/liter, most preferably from about 2 to about grams/liter.

It is usually required that the culture medium contains such nutrients for the microorganism as assimiable carbon sources, digestible nitrogen sources and preferably inorganic substances, vitamins, trace elements, other growth promoting factors, etc. L-sorbose per se can serve as the carbon source, but it is preferred to utilize other substances as carbon sources. Among the substances which can be utilized as carbon sources are included starch, cane sugar, lactose, dextrin, glycerol, maltose, etc. They can be employed at a concentration of from about 1 g./l. to about 10 g./l. As the nitrogen sources, there may be used various organic or inorganic substances such as soybean meal, meat extracts, peptone, casein, yeast extracts, corn steep liquor, urea, nitrates, ammonium salts, etc. As the inorganic nutrients, for example, potassium phosphates, magnesium sulfate, ferrous and ferric chlorides, calcium carbonate, etc., are usually employed. As the constituents of the medium vary also with the kind of microorganisms to be employed. it is preferable to choose a proper medium case by case.

In the case where after cultivation, the whole culture, i.e., the cells collected from the culture are brought into contact with the L-sorbose cultivation of the microorganisms is carried out under similar conditions described above. Substances mentioned above can, if desired, also be used for nutrients for this cultivation.

On the other hand, no nutrients need be present in cultivating the microorganisms. The whole grown culture is then utilized to convert L-sorbose to L- sorbosone. This conversion can be simply carried out in an aqueous medium under submerged conditions utilizing a pH of from about 5 to 9. In this'conversion, no additional nutrients need be present.

Generally, from about 1 to 3 days culture is preferable for obtaining the most effective cells for the conversion of L-sorbose to L-sorbosone. In one case, L-

.sorbose or its aqueous solution is added to the cultured .medium to make its final concentration from about 1 g./l. to about 200 g./l. The mixed solution may be incubated for about I to days under the same conditions as discussed above. In another case, the cells may be collected by centrifugation from the cultured broth and resuspended in an aqueous medium at a pH of from about 5 to 9. Then L-sorbose is added in the same way as mentioned above. The succeeding incubation may be effected under similar conditions as those described above.

On the other hand, when cell free extracts from the whole grown culture are utilized, these cell free extracts can be utilized to convert L-sorbose to L- sorbosone by treating L-sorbose with the cell free extracts in an aqueous medium at a pH of from 5 to 9. In this case, no nutrients need be present.

Where the whole cells or the cell free extract are utilized to convert L-sorbose to L-sorbosone, this conversion is generally carried out with no additional nutrients being present. However, if desired, any of the conventional nutrients such as those mentioned hereinbefore may be added to the aqueous fermentation medium containing either the whole grown cells or the cell free extract. Furthermore, in carrying out the microbiological conversion of L-sorbose with whole grown cells or with cell free extract, temperatures are not critical. However, it is generally preferred to utilize incubating temperatures of from to 40C. Generally, this microbiological conversion is carried out at a pH of from 5 to 9.

The reaction can bestopped by freezing the reaction medium, i.e., cooling the reaction medium to a temperature of 0C. or below to convert. Any temperature of 0C. or below can be utilized to stop the reaction. Generally, temperatures of from 5 to 50C. are utilized to stop the reaction. If desired, the L-sorbosone can be isolated by separating it from the reaction medium. Any coventional separation means can be utilized to carry out this isolation procedure. Among the preferred methods of isolation is by chromatography such EXAMPLE 1 Enzyme prepared from Pseudomonas putida Pseudomonas putida B (ATCC 21812) (20 ml. inoculum) were inoculated in 6 liter flasks containing 2 liters of a medium containing the following:

g./l. of distilled water Ammonium sulfate Sodium sulfate K HPO KH PQ, Sodium citrate CaCl 6H O FeCl 6H O ZnCl CuCl 2H O MnCl 4H O MgCl 6H O L-sorbose Glycerol The pH of the medium was adjusted to 7 and the medium was then autoclaved for 15 minutes at 15 psi. and a temperature of 151C. After autoclaving the cul tures were grown in the flasks by aeration on a shaker at approximately 280 RPM at 28C. for 8 hours. Cellpellets were obtained by centrifugation for 15 minutes at approximately 10,000 x gravity. The cell-pellets were pooled and resuspended in 500 ml. of cold 0.02M aqueous sodium phosphate buffer pH 7.0 and the suspension centrifuged again. The precipitates obtained were pooled and resuspended in 50 ml. of 0.02M aqueous sodium phosphate buffer pH 7.0 and processed twice through a French press 14,000 lb. per sq. inch. The resulting cell-extracts were centrifuged at approximately 27,000 X gravity for 30 minutes. The supernatents obtained after removal of debris were dialyzed twice against 0.02M-aqueous sodium phosphate pH 7.0 containing 1 X 10' M ethylenediamine tetraacetic acid (EDTA) and then dialyzed against buffer without EDTA. The resulting preparation designated dialyzed fraction 1, was used as given below.

Reaction mixtures were prepared containing in a total volume of 1.0 ml. of following:

Sodium phosphate, pH 7.0 40 p. mole L-sorbose 2.95 X 10 p. mole Dialyzed fraction 1 l5 mg. (protein) C-l'abellcd L-sorbose (l uCi; sp. act: 34 mCi/mM).

L-sorbosone (R,= 0.50). are clearly separated by this system. The developed strips (5.0 X 5 cm were air dried and then cut along the width into 1 cm segments. The folded segments were placed in vials for radioassays using a scintillation counter. Authentic samples of L-sorbosone and L-sorbose used as markers were located on the developed chromatog rams by spraying with a silver nitrate solution or by location of the radioactive authentic markers through radioassays counted in a scintillation counter. From the counts, the weight percent of L-sorbose converted to L-sorbosone were determined as given in the table below:

EXAMPLES 2 through 5 The organisms listed in Table 2 were grown in ether Medium 1 or Medium 2. The composition of Medium l, which was adjusted to a pH of 7, was as follows:

Grams/Liters of Distilled Water Glucose 5 Yeast Extract 5 Beef Extract 5 Difco bacto Peptone" 5 The composition of Medium 2 was as follows:

"A mixture of amino acids and peptides. A mixture of amino acids and peptides formed by the enzymatic hydrolysis of casein.

lnnoculations were p'erfomred by transferring a loopfull of each culture fromagar slants into 100 ml. of one of the two media in 500 ml. flasks. All cultures were grown with aeration on shakers (approximately 250 RPM) for 24 hours at 35C. with the exception that Serrariu murcescensyvas grown at 28C. for 48 hours. After the growth period, each culture was split into equal parts and centrifuged at approximately 10,000 times gravity for 15 minutes, at 4C. Each of the precipitates of the organisms was washed once with a buffer, i.e., either cold 0.02M aqueous sodium phosphate (pH 7.2) or cold 0.02M aqueous potassium phsophate (pH 7.2). The resulting buffered mixtures were then centrifuged as above. The precipitates were resuspended in 5 ml. of either of the buffers. Cell-extracts were prepared from each of the suspensions as in Example 1 except that only one passage through the French press was utilized. I

in addition, all extracts so prepared were dialyzed at 4C. either twice against 0.02M aqueous sodium phosphate buffer (pH 7.2) containing 1 X 10 M ethylene diamine tetraacetic acid EDTA followed by twice against the same buffer devoid of EDTA or twice against 0.02M potassium phosphate buffer containing 1 10 M EDTA buffer containing 1 X 10" M EDTA which was followed by twice against the last buffer devoid of EDTA to obtain the resulting cell-free extract preparation. The use of potassium phosphate or sodium phosphate is indicated in the following table:

Reaction mixtures containing each of the cell-free extracts prepared above were prepared with a volume of 1 ml. and a pH of 7.0 and containing the following ingredients:

L-sorbose l p. Ci Cell-free Extract 1 0.5 ml. Aqueous sodium or potassium phosphate umoles "'C -Labcl1ed L-sorbnse (72 mCi/mMolc:)

To these reaction mixtures there was added L- sorbosone at difficient concentrations as given in the table below. The reaction mixtures were incubated aerobically for 6 hours at 30C. The reactions were terminated by freezing at 20C. Analysis for L-sorbosone was carried out by the method given in Example 1. The results are given in Table 2. [n this table Bacillus s.p. was the strain TA.

Table 2 Conversion of L-Sorbose to L-Sorbosone Example Organism No. Grown lon 1n Percent Conversion of L-Sorbose in Media Cell-Free to L-Sorbosone based upon Extract L-Sorbosone added reaction medium 1 Serratia s.p. ATCC 93 1 Potassium 1.9 1.4 2 Serratia s.p. ATCC 93 2 Sodium 1.0 0.6

3 Bacillus s.p. ATCC 27860 1 Potassium 0.5 4 Bacillus s.p. ATCC 27860 2 Potassium 0.2 0.6 5 Sarcina lutea ATCC 9349 2 Potassium 0.4 0.4 6 Streptomyces ATCC 3313 2 Sodium 0.3 0.3

cellulosae 7 Serratia ATCC 27857 2 Sodium 0.3 0.1 marcescens 8 Aerobacter ATCC 27858 2 Potassium 1.1 1.0

aerogenes 9 Aerobacter ATCC 27858 2 Sodium 3.3 3.7

- aerogenes 10 Aerobacter ATCC 27858 1 I Sodium 2.9 1.5

aerogenes 'pmoles of L-Sorhosone added to the reaction mixture.

rwlaszutilizedrthe c'ation wa sjpotassiurn and 2.2 u moles f L-sorboson'was'added to the reaction mixture. The

lu rn) were inoculated in 6 liter flaskscontaining 2 liters of a medium which contains the ingredients and the amounts set forth in Example 1.

The pH of the medium was adjusted to 7 and the medium was then autoclaved for minutes at '15 psi. and a temperature of 151C. After autoclaving, the cultures were grown in the flasks by aeration on a shaker at approximately 250 RPM at 28C. for 18 hours. Cells were harvested by centrifugation for 15' minutes at approximately 10,000 X gravity. The cells were pooled and resuspended in 500 ml of.cold'0.02M aqueous sodium phosphate buffer pH 7.0 and the suspension centrifuged again. The washed cellsobtainedwere pooled and resuspended in 50 ml. of 0.02M aqueous sodium phosphate buffer pH 7.0 and processed twice through a French press 14,000 lb. per sq. and extracts centrifuged at 27,000 X gravity for 30 minutes. The resulting supernatent, i.'e. cell free extract was centrifuged at approximately 160,000 X gravity for 90 minutes. The supernatent obtained was designed as the soluble fraction; it was centrifuged again at high speed and the precipitate was discarded.

Reaction mixtures were prepared containing in a total volume of 1.0 ml. the following:

Sodium phosphate. pH 7.0 50 a mole L-sorbose 7 1.4' X 10- p. mole Soluble fraction 9.4 mg.

C-lahelled L-sorhose (72'MCi/mM).

carried out by paper chromatography utilizing the method described in Example 1. From the counts, the moles of L-sorbose converted to L-sorbosone was determined as given in the table below:

Table 3 Reactions Cofactor oft tal counts in 'fC; sorbosone t I 1 None 03 II NAD 0.5 111 NADP 0.2

EXAMPLE 7 By the procedure given in Example 2 through 5, utilizing Paecilomyces varioti (ATCC 26820) and Mycobacterium phlei (ATCC 355), L-sorbose was converted to L-sorbosone.

In the case of Mycobacterium phlei, the medium 2 {percent-conversion of L-sorbose to L-sorbosone was 0.3%.

1 In thecase of Paecilomy c' 's v a iiioti, the medium 1 was utilized, the cation waspoptassium and2.2 [1.1110168 of L-sorbosone was added to thereactiommedium. The

percent conversiori of L-sorbose toL -sorbosone was We claim: i

1. A method for producing L-sorbosone from L- sorbose which comprises pre-culturing under submerged aerobic conditionsat a temperature of from 20 to 45C. and a pH of from 5 to 9, a microorganism selected from the group consisting of:

Paecilomyces;

Mycobacterium;

Pseudomonas;

Serratia,

Bacillus;

Aerobacter;

Streptomyces; I

Gluconobacter;

Sarcina; and

v Acinetobacter, I

in an aq ue o us medium to produce: cells containing an enzyme system capable of convert in'giL-sorbose to L- sorbo'sone, incubating under submerged aerobic conditions at a temperature of from 20 to 45C. and a pH of from 5 to 9;said cells with a medium containing L- Q b Q o'nvsr t e L-sotbose to L-sorbqs n an recovering L-sorbosone from said media. v

2. The process of claim 1 wherein said microorganism is Pseudomortas putida. 1

3. The process ofclaim- 1 wherein said microorganism is Aeorbacter aerogenes.

4. A process for the preparation of -L-sorbosone which comprises pre-culturing, under submerged aerobic conditions at a temperature of from 20 to 45C. and a pH of from, 5,t0 9,ja microorganism selected from the group consisting of:

seudomonas;

Serratia;

Bacillus;

I Aerobacter;

Streptomyces;

Sarcina;

Mycobacterium;

Acinetobacter;

Gluconobacter; and v Paecilomyces, I in an aqueous medium to produce zzells containing an enzyme system capable of convertiirig L-sorbose to L- sorbosone, extracting said enzymf system from said cells and incubating this system, under submerged aerobiclconditions ,at a temperature of from 20 to 45C.

and apH of'from 5-10 9, {with a L-sorbose containing medium to convert'lthe L-sorbose in the medium to L- sorbosone. r I

5. The process of claim 4 wherein said micr0organism is .Pseudomonas putida.

6. The process of claim 4 wherein said microorgan- 

1. A METHOD FOR PRODUCING L-SORBOSONE FRON L-SORBOSE WHICH COMPRISES PRE-CULTERING UNDER SUBMERGEG AEROBIC CONDITIONS AT A TEMPERATURE OF FROM 20* TO 45*C. AND A PH OF FROM 5 TO 9, A MICROORGANISM SELECTED FROM THE GROUP CONSISTING OF: PAECILOMYCES, MYCOBACTERIUM, PSEUDOMONAS, SERRATIA, BACILLUS, AEROBACTER, STREPTOMYCES, GLUCONOBACTER, SARCINA, AND ACINETOBACTER, IN AN AQUEOUS MEDIUM TO PRODUCE CELLS CONTAINING AN EZYME SYSTEM CAPABLE OF CONVERTING L-SORBOSE TO L-SORBOSONE, INCUBATING UNDER SUBMERGED AEROBIC CONDITIONS AT A TEMPERATURE OF FROM 20* TO 45*C, AND A PH OF FROM 5 TO 9, SAID CELLS WITH A MEDIUM CONTAINING L-SORBOSE TO CONVERT THE L-SORBOSE TO L-SORBONE AND RECOVERING L-SORBONE FROM SAID MEDIA.
 2. The process of claim 1 wherein said microorganism is Pseudomonas putida.
 3. The process of claim 1 wherein said microorganism is Aeorbacter aerogenes.
 4. A process for the preparation of L-sorbosone which comprises pre-culturing, under submerged aerobic conditions at a temperature of from 20* to 45*C. and a pH of from 5 to 9, a microorganism selected from the group consisting of: pseudomonas; Serratia; Bacillus; Aerobacter; Streptomyces; Sarcina; Mycobacterium; Acinetobacter; Gluconobacter; and Paecilomyces, in an aqueous medium to produce cells containing an enzyme system capable of converting L-sorbose to L-sorbosone, extracting said enzyme system from said cells and incubating this system, under submerged aerobic conditions at a temperature of from 20* to 45*C. and a pH of from 5 to 9, with a L-sorbose containing medium to convert the L-sorbose in the medium to L-sorbosone.
 5. The process of claim 4 wherein said microorganism is Pseudomonas putida.
 6. The process of claim 4 wherein said microorganism is Aerobacter aergenes. 